Abstract

891

Our previous work demonstrated that inactivation of a single p21 allele (ie Apc+/−,p21+/−) was sufficient to augment and accelerate Apc initiated tumor formation (Yang et al, Cancer Res., 61, 565, 2001) and that inactivation of only one p21 allele was also sufficient to abrogate tumor inhibition by sulindac, a non-steroidal anti-inflammatory drug (Yang et al, Cancer Res., 61, 6297, 2001). To investigate the underlying mechanism, we used quantitative real time PCR, western blots and tissue microarray to analyze altered expression of the p21WAF1 gene, the functionally linked gene c-myc and the down-stream target of c-myc, cdk4. We found that sulindac could induce p21 expression in both normal mucosa and tumors from p21 wild-type, Apc+/− mice, that the induction of p21 inhibited c-myc expression at both the mRNA and protein level, and that this in turn inhibited cdk4 expression. However, in the p21 heterozygote, Apc+/− mice, sulindac could not induce p21, and thus did not inhibit c-myc and cdk4 expression. Therefore, we investigated whether the remaining wild-type p21 allele in the Apc+/−, p21+/− mice was in fact silenced by methylation. Genomic DNA was isolated from the normal mucosa and tumor tissues, converted with bisulfite, then methylation-specific PCR was performed and the PCR product was cloned into a pCR4-vector and sequenced. As expected, the two wild-type p21 alleles were unmethylated in the normal mucosa of p21+/+, Apc+/− mice, but the remaining wild-type allele became hypermethylated in the normal mucosa and in the tumors of the p21+/− mice. This suggested that although the p21+/− mice retained a wild-type allele, this allele appeared to be functionally inactivated by hypermethylation. Hence, the augmentation of tumor formation in Apc+/−,p21+/− mice likely results from complete loss of p21 expression even though a wild-type p21 allele is retained. Furthermore, the inability of sulindac to inhibit tumor formation in Apc+/−,p21+/− mice is likely due to the inability to induce expression of the wild-type, but methylated, remaining p21 allele.